Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model

Abstract

Murine studies have shown that immunological targeting of fibroblast activation protein (FAP) can elicit protective immunity in the absence of significant pathology. Fibroblast activation protein is a product overexpressed by tumor-associated fibroblasts (TAF) and is the predominant component of the stoma in most types of cancer. Tumor-associated fibroblasts differ from normal adult tissue fibroblasts, and instead resemble transient fetal and wound healing-associated fibroblasts. Tumor-associated fibroblasts are critical regulators of tumorigenesis, but differ from tumor cells by being more genetically stable. Therefore, in comparison to tumor cells, TAF may represent more viable therapeutic targets for cancer immunotherapy. To specifically target TAF, we constructed a DNA vaccine directed against FAP. This vaccine significantly suppressed primary tumor and pulmonary metastases primarily through CD8(+) T-cell-mediated killing in tumor-bearing mice. Most importantly, tumor-bearing mice vaccinated against FAP exhibited a 1.5-fold increase in lifespan and no significant pathology. These results suggest that FAP, a product preferentially expressed by TAF, could function as an effective tumor rejection antigen.

Figure 1

Effects of fibroblast activation protein (FAP) expression and vaccination on tumor growth and lifespan of tumor‐bearing mice. (a) Western blot analysis of FAP expression and (b) dipeptidyl peptidase (DPP) activity in untransfected, PVITRO2‐neo‐mcs (pVector)‐transfected and PVITRO2‐neo‐mcs‐FAP (pFAP)‐transfected CT26 cells. pFAP‐transfected CT26 cells exhibited significantly increased FAP expression and DPP activity compared with controls (*P < 0.05). (c) BALB/c mice (n = 5) were challenged by s.c. injection with 1 × 10⁵ untransfected, pVector‐transfected or pFAP‐transfected CT26 cells. Mice challenged with pFAP‐transfected CT26 cells (pFAP‐CT26 mice) exhibited a significant increase in tumor volume in comparison with control groups (*P < 0.05) (c). (d–e) Seven days after the last of six vaccinations administered at 1‐week intervals, BALB/c mice (n = 7 per group) were challenged with a lethal dose of 1 × 10⁵ CT26 cells delivered by s.c. injection. (d) A significant decrease in tumor volume (*P < 0.05) was present in pFAP‐immunized mice compared with control mice immunized with pVector or normal saline (NS). (e) A significant increase in survival occurred in pFAP‐immunized mice in comparison with control mice (P < 0.05, log‐rank test). Experiment was performed in duplicate for each group. Error bars indicate mean ± SD.

Figure 2

Inhibition of lung metastasis in mice immunized with fibroblast activation protein (FAP). BALB/c mice were immunized with PVITRO2‐neo‐mcs‐FAP (pFAP), PVITRO2‐neo‐mcs (pVector) or normal saline (NS) six times at 1‐week intervals, injected i.v. with 1 × 10⁵ CT26 cells and killed on day 23 after injection. (a) Representative images of mouse lungs. (b) Average lung weights. Lungs from pFAP‐immunized mice were significantly smaller than those of the control groups (*P < 0.05). Error bars indicate mean ± SD. (c) Percentage of lung surface covered by fused metastases.

Figure 3

Antitumor effects of the fibroblast activation protein (FAP) vaccine are mediated by CD8⁺ T cells. (a–b) T cells (1 × 10⁷) obtained from BALB/c mice immunized with PVITRO2‐neo‐mcs‐FAP (pFAP), PVITRO2‐neo‐mcs (pVector) or normal saline (NS) were injected into syngeneic mice (n = 6) 1 day after inoculation with 1 × 10⁵ CT26 cells. Suppression of tumor growth (*P < 0.05) (a) and a significant increase in survival (P < 0.05, log‐rank test) (b) were observed in mice injected with T cells from pFAP‐immunized mice compared with controls. (c–e) T lymphocytes from mice immunized with pFAP, pVector or NS were incubated with pFAP‐transfected (c), pVector‐transfected (d) or untransfected CT26 (e) cells at the indicated effector/target ratios, and in vitro cytotoxicity was measured as ⁵¹Cr release. T cells isolated from mice immunized with pFAP exhibited increased cytotoxicity against FAP‐positive target cells (P < 0.05), but not against FAP‐negative target cells. A representative graph of triplicate samples from the experiment is shown. (f) CD4⁺ or CD8⁺ T lymphocytes or natural killer (NK) cells were depleted using corresponding antibodies in immunized tumor‐bearing BALB/c mice (n = 6 per group), and tumor volume was assessed. Significant suppression of tumor growth was observed in pFAP, pFAP/anti‐CD4⁺ and pFAP/anti‐NK mice, but not pVector, NS or pFAP/anti‐CD8⁺ mice (*P < 0.05). No significant difference was observed between pFAP and pFAP/anti‐CD4⁺ or pFAP/anti‐NK mice. (g–i) Depletion of CD8⁺ T lymphocytes inhibited the antitumor activity of the pFAP vaccine in the CT26 lung metastasis model (n = 5 per group). (g) Representative images of mouse lungs. (h) Average lung weights. The lungs from pFAP, pFAP/anti‐CD4⁺ and pFAP/anti‐NK mice were significantly smaller than that of pFAP/anti‐CD8⁺ mice and the control groups (*P < 0.05). No significant difference in lung weight was observed between pFAP, pFAP/anti‐CD4⁺ or pFAP/anti‐NK mice. Error bars indicate mean ± SD. (i) Percentage of lung surface covered by fused metastases.

Figure 4

Immunohistochemical characterization of tumor tissues in immunized mice. Tumor sections from mice immunized with normal saline (NS) (a,d,g,j,m), PVITRO2‐neo‐mcs (pVector) (b,e,h,k,n) or PVITRO2‐neo‐mcs‐FAP (pFAP) (c,f,i,l,o), or colon tissue sections from control mice (p) were stained with HE (a–c), anti‐CD8 antibodies (d–f), with the TUNEL assay (g–i), with antibodies directed against fibroblast activation protein (FAP) (j–k,p), and with Sirius Rose BB to stain for stromal collagen (m–o). In comparison with mice immunized with NS or pVector, increased lymphocyte and CD8⁺ T cell infiltration (black and white arrows, respectively), increased numbers of apoptotic cells (white arrows), and decreased FAP and collagen expression (red and black arrows, respectively) were observed in tumors from pFAP‐immunized mice. (a–c,j–o,p) ×40 magnification; (d–i) ×20 magnification.